Biology of Spirochetes  


  HEADDr PICARDEAU Mathieu / mpicard@pasteur.fr
  MEMBERSResearcher PICARDEAU Mathieu IP Assistant Professor, PhD
Research Engineer BOURHY Pascale IP Engineer, PhD Deputy manager of the NRC for Leptospira
Research fellows LOURDAULT Kristel Master 2 student / CERQUEIRA Gustavo PhD student / AVIAT Florence postdoc student
Other staff ZININI Farida IP Technician / COUEILLE Solange IP Secretary / MOREL Viviane IP Technician / CLEMENT Sabine IP Technician


  Annual Report

Spirochetes are the causative agents of several important animal and human diseases such as Lyme disease and leptospirosis. The unit is composed of a research group on Leptospira and the National Reference Center for Leptospira (also WHO collaborating Center for Leptospira ).

In 2008, the main research activities included:

i) Comparative genomics between saprophytic and pathogenic Leptospira

In collaboration with the Genomic Platform of the Pasteur Institute, we sequenced the genome of the saprophyte Leptospira biflexa which is our model bacteria. Indeed, numerous tools for genetic manipulation of this saprophytic strain have been developed by our group in the recent years, including random and targeted mutagenesis. We have started the comparison of the genetic content between L. biflexa and pathogenic species that have been already sequenced. Comparative genomics reveals clues on the life-styles of Leptospira in the environment and in the infected host.

ii) First evidence of targeted mutagenesis in pathogenic Leptospira

Leptospiral immunoglobulin- like repeat (Lig) proteins were previously identified as a putative Leptospira virulence factor. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans, wherein a spectinomycin resistance gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with ligB mutant was able to induce persistent colonization of the kidneys. Finally LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provides the first evidence for site-directed homologous recombination in pathogenic Leptospira. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and development of acute disease manifestations or persistent renal colonization (Croda et al. 2008).

iii) Characterization of biofilm formation in Leptospira spp.

Leptospires exist as saprophytic organisms that are exclusively aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, that are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochetes were able to form a biofilm, i.e surface-associated cells enclosed in a polymer matrix of their own synthesis. We have demonstrated, by electron and confocal laser-scanning microscopy, that L. biflexa and L. interrogans strains can form biofilms in vitro. The biofilm formation in vitro by Leptospira strains was also investigated on abiotic surfaces (polystyrene microtiter plates) by measuring absorbance after staining with crystal violet. Using this method, we were able to show that L. biflexa mutant strains form less extensive biofilms in vitro than the wild-type strain (Ristow et al. 2007).

iv) Systematic mutagenesis of the genome of the pathogen L. interrogans

We recently provided the first evidence of gene transfer in a pathogenic Leptospira strain, involving the transposition of a transposon of eukaryotic origin (Bourhyet al. 2005). We used this methodology in L. interrogans serovar Lai strain Lai 56601, a pathogenic strain which is entirely sequenced (Ren et al. 2003) and relatively transformable, to construct a library of random mutants. The L. interrogans genome contain approximately 3400 predicted coding regions (excluding transposases and pseudogenes), of which half were assigned no biologic role, whereas the remaining half were assigned roles that await experimental validation. The insertion sites of the transposon has been determined in approximately 300 random mutants of L. interrogans strain Lai. We also have generated libraries of random mutants in other pathogenic strains, then generating approximately 1000 defined mutants with characterized transposon insertion points (Murray et al. 2009). Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

Keywords: Spirochetes, Leptospira, epidemiology, genetics, virulence factors



  Publications

Bourhy, P., H. Louvel, I. Saint Girons, and M. Picardeau. 2005. Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon. J. Bacteriol. 187:3255-3258. PMID: 15838053

Salaun, L., F. Merien, S. Gurianova, G. Baranton, and M. Picardeau. 2006. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J. Clin. Microbiol. 44:3954-3962. PMID: 17088367

Ristow, P., P. Bourhy , F.W. McBride, C.P. Figueira, M. Huerre, P. Ave, I.S. Girons, A.I. Ko, and M. Picardeau. 2007. The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathog. 3: 0894-0903. PMID: 17630832

M. Picardeau, D.M. Bulach, C. Bouchier, R. L. Zuerner, N. Zidane, P. J. Wilson, S. Creno, E. S. Kuczek, S. Bommezzadri, J. C. Davis, A. McGrath, D. Roche, M. J. Johnson, C. Boursaux-Eude, T. Seemann, Z. Rouy, J. I. Rood, A. Lajus, J. K. Davies, C. Médigue, and B. Adler (2008). Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis PLoS ONE 3(2):e1607.

J. Croda, C.P. Figueira, E.A. Jr Wunder, C.S. Santos, M.G. Reis, A.I. Ko, and M. Picardeau (2008). Targeted mutagenesis in pathogenic Leptospira: Disruption of the ligB gene does not affect virulence in animal models of leptospirosis.Infect. Immun.76:5826-33.



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Activity Reports 2009 - Institut Pasteur
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